Validamycin c, d, e and f antibiotics

ABSTRACT

Novel antibiotics, termed validamycins C, D, E and F, and acid salts thereof, are prepared by cultivating a validamycin-producing strain of microorganism of the genus Streptomyces, and optionally converting the free base to its acid salt.

The present invention relates to validamycins C, D, E and F, andvalidoxylamines A and B, and acid salts thereof, obtained by cultivatinga validamycin-group-producing strain of microorganism belonging to thegenus Streptomyces.

In search of new antibiotic substances, the present inventors isolated alarge number of soil microorganisms and studied their metabolites. Theresearch work led to the findings that certain soil microorganisms arecapable of producing validamycins C, D, E and F, and validoxylamines Aand B, that those microorganisms belong to the genus Streptomyces andthat it is possible to obtain said antibiotics by cultivating thosemicroorganisms so that the said antibiotics may be accumulated in theculture medium.

This invention is the culmination of the foregoing discoveries. Thus,the invention relates to validamycins C, D, E and F, and validoxylaminesA and B, which are prepared by cultivating a validamycin-group producingstrain of microorganism belonging to the genus Streptomyces so that theantibiotic may be produced; accumulating the validamycins C, D, E and F,and validoxylamines A and B in the fermented broth; and recovering theantibiotics so accumulated from the fermented broth.

Furthermore, it is found unexpectedly that these validamycins C, D, Eand F, and validoxylamines A and B show an excellent controlling effectagainst plant diseases, for example, the sheath blight of the rice plantby application in vivo, although they show no antimicrobial potency invitro against bacteria and fungi. Further study has revealed that thesevalidamycins C, D, E and F, and validoxylamines A and B have a peculiarcharacteristic that only when the plant or soil is treated therewith,they show a strong disease-controlling effect.

Furthermore, the validamycins C, D, E and F, and validoxylamines A and Bhave been found to be very effective not only against the sheath blightof the rice plant but also against other plant diseases such as the stemrot of rice plant and the damping off and sclerotial blight ofvegetables, flowering plants and lumber seedlings. In addition, thevalidamycins C, D, E and F, and validoxylamines A and B aresubstantially harmless to men and beasts, as well as to fish, and evenwhen applied in a high concentration, they do not substantially affectthe germination, growth, yield and other features of useful plants.

The principal object of this invention is to provide a process forpreparing validamycins C, D, E and F, and validoxylamines A and B.

Another object is to provide new validamycins C, D, E and F.

Further object is to provide a fungicide for combating fungal plantdiseases showing no substantial phytotoxicity and substantiallynon-toxic against both human beings and animals as well as fish.

Other objects will be apparent from the description detailed hereinafterin this specification.

Validamycins C, D, E and F, and validoxylamines A and B (hereinafterreferred to sometimes as only "the antibiotics") are obtained bycultivation of validamycin-group producing strain belonging to the genusStreptomyces, so long as it is capable of producing these antibiotics.For example, the strain which was isolated from the soil collected inAkashi City, Hyogo Prefecture, Japan by the present inventors and namedStreptomyces hygroscopicus var. limoneus (ATCC No. 21431 and ATCC No.21432) as well as its related strains, may be employed to particularadvantage. These microorganisms are disclosed in co-pending applicationSer. No. 871,899, now U.S. Pat. No. 3,754,083.

Some of the microbiological and cultural characteristics of abovementioned Streptomyces hygroscopicus var. limoneus are shown below. Inthe following descriptions of the cultural characteristics, "Rdg." meansthe color name according to Ridgeway's "Color Standard and colorNomenclature."

1. Morphological characteristics

The aerial mycerium of this microorganism is simply branching andspore-bearing hyphae form spiral.

The conidium is ovoid or rectangular, ranging from 1.0-1.3 μ × 1.0-1.5 μand has a smooth surface. This microorganism has no sclerotia,flagellated spores and sporangia.

2. Cultural characteristics

Unless otherwise specified, the characteristics described below arethose observed upon cultivation at 28° C. When the microorganism wasgrown at temperatures other than 28° C, the particular culturaltemperatures are indicated in parentheses.

a. Czapek's agar

Growth: Colorless, folded.

Reverse: Raw Sienna (Rdg., III, 17-i) to Sudan Brown (Rdg., III, 15-k).

Aerial mycelium: Tilleul Buff (Rdg., XL, 17'"-f) to Light Buff (Rdg.,XV, 17'-f), partially Mouse Gray (Rdg., LI, 15""') along the peripheryof the colony.

Soluble pigment: Yellow with a faint brownish tinge.

b. Glucose Czapek's agar

Growth: Colorless to Sulphin Yellow (Rdg., IV, 21-i), folded.

Reverse: Raw Sienna.

Aerial mycelium: Tilleul Buff to Massicot Yellow (Rdg., XVI, 21'-f),partially Light Olive Gray (Rdg., LI, 23""'-d) along the periphery ofthe colony. Soluble pigment: Yellow with a faint brownish tinge.

c. Glycerin Czapek's agar

Growth: Colorless to Orange Citrine (Rdg., IV, 19-k), folded.

Reverse: Raw Sienna.

Aerial mycelium: Tilleul Buff to Massicot Yellow, partially Light OliveGray.

Soluble pigment: Yellow with a faint brownish tinge.

d. Glucose asparagine agar

Growth: Colorless.

Reverse: Old Gold (Rdg., XVI, 19'-i) to Antimony Yellow (Rdg., XV,17'-b) to Cinnamon Brown (Rdg., XV, 15'-k).

Aerial mycelium: Light Olive Gray to Mouse Gray, with yellow patches andblack moist areas.

Soluble pigment: Light Brown.

e. Calcium malate agar

Growth: Primuline Yellow (Rdg., XVI, 19')

Reverse: Primuline Yellow.

Aerial mycelium: Scarce at first, but Tilleul Buff to Light Olive Graylater.

Soluble pigment: Pale Yellow.

f. Starch agar

No growth.

g. Modified starch agar with the following components

    ______________________________________                                        Soluble starch        1%                                                      Potassium hydrogen phosphate                                                                        0.3%                                                    Calcium carbonate     0.3%                                                    Magnesium sulfate     0.1%                                                    Ammonium sulfate      0.2%                                                    Sodium chloride       0.05%                                                   Agar                  2%                                                      ______________________________________                                    

Growth: Colorless to Barium Yellow (Rdg., XVI, 23'-d).

Reverse: Deep Colonial Buff (Rdg., XXX, 21"-b) to Snuff Brown (Rdg.,XXIX, 15"-k).

Aerial mycelium: Cartridge Buff (Rdg., XXX, 19"-f) to Mouse Gray, withblack moist areas.

Soluble pigment: Light Brown Hydrolipio of starch was observed.

h. Tyrosine agar

Growth: Colorless to Strontian Yellow (Rdg., XVI, 23').

Reverse: Pale Ochraceous Buff (Rdg., XV, 15'-f) to Light Ochraceous Buff(Rdg., XV, 15-d).

Aerial mycelium: None.

Soluble pigment: None.

i. Yeast extract agar

Growth: Colorless, folded.

Reverse: Cream color (Rdg., XVI, 19'-f).

Aerial mycelium: White.

Soluble pigment: Light Brown.

j. Nutrient agar (37° C)

Growth: Colorless.

Reverse: Colorless.

Aerial mycelium: None.

Soluble pigment: None.

k. Glucose Nutrient agar (37° C)

Growth: Colorless, wrinkled.

Reverse: Cartridge Buff to Pale Ochraceous Buff.

Aerial mycelium: None.

Soluble pigment: None.

l. Nutrient broth (37° C)

Growth: Colorless surface growth, and colorless flocculent growth in thebottom.

Aerial mycelium: None.

Soluble pigment: None.

m. Glucose Nutrient broth (37° C)

Growth: Surface growth, Cartridge Buff, and colorless flocculent growthin the bottom.

Aerial mycelium: None.

Soluble pigment: None.

n. Potato plug

Growth: Colorless to Pale Ochraceous Buff.

Aerial mycelium: Tilleul Buff to Mouse Gray. The plug turns to SayalBrown (Rdg., XXIX, 15"-i).

o. Carrot plug

Growth: Colorless.

Aerial mycelium: White to Mouse Gray. The plug turns to Cinnamon Rufous(Rdg., XIV, 11'-i) to Cinnamon Brown.

p. Celulose

Growth: Chartreuse Yellow (Rdg., XXXI, 25"-d) to Reed Yellow (Rdg., XXX,23"-b).

Aerial mycelium: Mouse Gray.

Soluble pigment: Pale Yellow.

q. Gelatin (25° C)

Growth: Very poor.

Aerial mycelium: None.

Soluble pigment: None.

Gelatine is liquefied slightly.

The same is true with nutrient gelatin.

r. Whole egg (37° C)

Growth: Colorless.

Aerial mycelium: None.

Soluble pigment: None.

s. Litmus milk (37° C)

Growth: Surface growth, Cream color to Seashell Pink (Rdg., XIV, 11'-f).

Aerial mycelium: None.

The medium is weakly coagulated, then, peptonized to turn to Army Brown(Rdg., XL, 13"'-i) and becomes weakly acidic.

Loffler's medium (37° C)

Growth: Naples Yellow (Rdg., XVI, 19'-d) at first and Light Buff later.

Aerial mycelium: None.

Soluble pigment: None.

No liquefaction.

u. Peptone glucose agar

Growth: Charteruse Yellow

Reverse: Honey Yellow (Rdg., XXX 19").

Aerial mycelium: Thin, Cream Buff (Rdg., XXX 19"-d).

Soluble pigment: Yellow with a brownish tinge.

v. Plain Agar

Growth: Scarce and colorless growth into substance of medium.

Reverse: Colorless to Mouse Gray.

Aerial mycelium: Scarce, Tilleul Buff to Mouse Gray.

Soluble pigment: None.

3. Physiological properties

a. Temperature and pH range.

no growth at 10° C and at 50° C on Bennett's agar or glucose aspargineagar under aerobic conditions. Growth occurs at 15°-45° C, and bettergrowth at 37°-45° C. No growth when medium is pH 4. While growth occursat pH 5-10, the optimum range is pH 6-7.

b. Gelatin: Slightly liquefied.

c. Starch: Hydrolyzed. Dia. of hydrolyzed area/dia. of colony=33mm/8mm.

d. Tyrosinase reaction: Negative.

e. Litmus milk: Peptonized. Coagulation, doubtful.

f. Reduction of nitrate: Negative.

g. Hydrolysis of cellulose: Negative

h. Chromogenicity: Negative.

i. Product: Validamycin-group antibiotics.

4. Utilization of carbon sources

Table 1 shows the utilization of carbon sources by the present strain asexamined by the method of Pridham et al. (Journal of Bacteriology 56107-114(1948)).

                  TABLE 1                                                         ______________________________________                                        Utilization of carbon sources by Streptomyces                                 hygroscopicus var. limoneus                                                   ______________________________________                                        Erythritol  -      Inositol            ++                                     Adonitol    -      D-mannitol          ++                                     Sorbitol    +      Dulcitol            -                                      D-Xylose    ++     Trehalose           ++                                     L-Arabinose ++     Salicin             -                                      L-Sorbose   -      Esculin             -                                      D-Galactose ++     Inulin              ++                                     Glucose     ++     Dextran             +                                      D-Fructose  ++     Mannose             ++                                     L-Rhamnose  ++     Starch              ++                                     Melibiose   ++     Glycerol            ++                                     Maltose     ++     Sodium acetate      +                                      Sucrose     ++     Sodium succinate    +                                      Lactose     ++     Sodium citrate      +                                      Raffinose   ++     Calcium 2-keto gluconate                                                                          -                                      ______________________________________                                         ++: well utilized;                                                            +: fairly utilized;                                                           -: not utilized.                                                         

Thus, the present strain shows monopodial branching, the tip of itsaerial mycelium being coiled. The conidia have a smooth surface. Itgives bright yellow to buff-colored growth on synthetic media,generally; produces no brown soluble pigment on protein-containingmedia.

The foregoing cultural characteristics were compared with thedescriptions given in "Bergey's Manual of Determinative Bacteriology",7th ed. (The Williams and Wilkins, 1957), S.A. Waksman's "TheActinomycetes", Vol. 2 (The Williams and Wilkins, 1962) and "RalphHutter's Systematik der Streptomyceten"(Es.Karugas, 1967), for instance.It was found that the strain resembles Streptomyces ambofaciens,Streptomyces platensis and Streptomyces hygroscopicus.

However, despite of the close resemblance in the color of bothvegetative and aerial mycelium, Streptomyces ambofaciens and the presentstrain differ from each other in that the former does not give blackmoist spots in the aerial mycelium, that it liquefies gelatin in amedium degree and gives yellow flocculent growth in the liquefied part.There also are difference between both organisms in carbon sourceutilization. Streptomyces platensis differentiates itself from thepresent strain in that the former produces a Deep Olive vegetativemycelium on Czapek's agar, with the reverse of the colony turning darkolive with the passage of time, that it gives cream to dull yellowishgrowth on starch agar with its aerial mycelium changing in color fromwhite to Mouse Gray with black patches. They are different from eachother in carbon source utilization. Comparison of the descriptions ofStreptomyces hygroscopicus with the cultural characteristics of thepresent strain shows that the present strain differentiates itself fromStreptomyces hygroscopicus in that the growth or the reverse of thecolony of the present strain shows the light yellow to buff on Czapek'sagar (inclusive of glucose Czapek's agar and glycerol Czapek's agar),glucose asparagine agar, calcium malate agar and other media, andproduces a yellowish white to yellow aerial mycelium on said Czapek'sagar media. However, many of the cultural characteristics of this straincoincide with the stable characteristics of Streptomyces hygroscopicusindicated by Tresner and Backus (Applied Microbiology, Vol.4, p.243,1956). Accordingly we identified the present strain as a variety ofStreptomyces hygroscopicus and designated it as Streptomyceshygroscopicus var. limoneus.

As a general trait of the genus Streptomyces, their microbiologicalcharacteristics are highly mutative and Streptomyces hygroscopicus var.limoneus is no exception to the rule. For example, its culturalcharacteristics and pattern of utilization of carbon sources aresusceptible to change, and there can be many mutants. Particularly, ofthis strain, mutants which have yellow aerial mycelia are easilyobtained. However, even those mutants may be employed in this inventionin so far as they possess the capacity to produce the validamycin-groupantibiotics. It does not matter, of course, if the mutants are inducedspontaneously or artificially. For purposes of this invention thesemutants are the full equivalents of the above-denominated strains.

In the culture medium employed in this invention, assimilable carbonsources, digestible nitrogen sources, inorganic salts and the like areincorporated. If required, there may be added trace elements such astrace nutrients, growth factors, precursors, etc. to the culture medium.The carbon sources which the validamycin-group producing strainassimilates include, among others, hydrocarbon, glucose, sucrose,molasses, starch, dextrin and glycerine. The nitrogen sources includesuch organic nitrogenous compounds such as meat extract, soybean flour,corn steep liquor, peptone, casein, etc., as well as such inorganicnitrogen compounds as nitrates and ammonium compounds, and any of themcan be employed to advantage.

While cultivation can be carried out by surface culture, it is moreusual to adopt the aerobic submerged culture. In the case of thesubmerged culture, the pH of the medium is preferably near neutral, andwhile growth occurs at the incubation temperature of 20° to 40° C, it ispreferable to maintain the medium within the range of about 23° to 37°C. The accumulation of the objective antibiotic completes in 4 to 7days.

The antibiotics do not inhibit the growth of bacteria and fungi in vitrobut only cause Pellicularia sasakii and its closely related fungi anabnormal branching (excessive branching, or branched hyphae become anumbel like form) on the tips of the hyphae. Therefore, the bioassay ofthe antibiotics should be conducted in the following manner.

Pellicularia sasakii is used as the test organism employing plain agaras the assay medium. The test organism is cultivated on a potato(sucrose) agar slant for 2 to 5 days, and the resulting culture is usedto inoculate at the center of a 9-cm Petri dish plate of modifiedPfeffer's medium, which is then incubated at 27° C for 2 days. By theend of this period, the mycelium will have spread over the entiresurface of the plate. The growth on the circumference about 3 to 3.5 cmin radius from the center is cut out with a cork borer and the agar discthus obtained is used as the inoculum.

A serial dilution series of agar plates containing varyingconcentrations of the antibiotics is prepared in the same manner as aconventional agar dilution method.

A glass disc of 8 mm in diameter and about 02.mm thick, is placed in thecenter of each of the aforementioned dilution series of agar plates, andthe agar disc inoculum is then placed on the glass disc. Afterincubation at 27° C for 40 hours, the result is evaluated. Naked-eyeexamination reveals that in Pellicularia sasakii the tip of hyphae oftest organism growing from the agar disk inoculum undergoes abnormalbranching. The term dilution unit means the value of maximum dilutionwhich produces such abnormal branching. The aqueous solution containing1000 γ/ml. of purified validamycins C, D, E and F, and validoxylamines Aand B shows 100, 10, 80000, 80000, 100 and 10 dilution unit/ml.,respectively.

Modified Pfeffer's medium, which is employed in this assay, has thefollowing composition.

    ______________________________________                                        Sucrose               3%                                                      L-asparagine          0.2%                                                    Ammonium nitrate      0.3%                                                    Potassium dihydrogen                                                           phosphate            0.1%                                                    Magnesium sulfate     0.1%                                                    Velsenol*             0.001%                                                   (Dow Chemical)                                                               Agar                  1.2%                                                     Adjusted to pH 7.                                                            ______________________________________                                    

Before using the medium, the following vitamins are added in thespecified amounts. (Weight per ml. of medium)

    ______________________________________                                        Thiamine            1 γ /ml.                                            Riboflavin          1 γ/ml.                                             Calcium pantothenate                                                                              1 γ/ml.                                             Niacin              1 γ/ml.                                             Biotin              0.005 γ/ml.                                         Folic acid          0.5 γ/ml.                                           Pyridoxine hydrochloride                                                                          2 γ/ml.                                             p-Aminobenzoic acid 0.5 γ/ml.                                           Cyanocobalamine     0.0002 γ/ml.                                        ______________________________________                                         *A chelate compound, iron sodium ethanol ethylene-diamine triacetate.    

When Streptomyces hygroscopicus var. limoneus is cultivated in the abovemanner, the antibiotics are produced and accumulated mainly in theliquid phase of the broth. Therefore, to recover these antibiotics, itis preferable to filter the broth and then recover the antibiotics fromthe resulting filtrate. When the microorganism employed is capable ofproducing only one of the antibiotics, the antibiotics can, of course,be collected from the culture broth in per se conventional manner. But,in case where the microorganism employed can produce simulataneously twoor more of them, they are concomitantly accumulated in the broth, sothat those antibiotics are collected from the broth, as a mixture ofthem or as an individual validamycins C, D, E or F, or validoxylamines Aor B.

For the purpose of isolating the antibiotics, means which areconventionally used to recover the metabolites of microorganisms fromtheir broths can be employed either singly or in combination. Thus, themeans include such techniques as filtration, concentration, ion-exchangechromatography with ion exchangers, adsorption chromatography on activecarbon, silica gel or alumina, gel filteration with Sephadex (Trade nameof Pharmacia) or Bio-Gel-P (Trade name of BIO. RAD Laboratories), theuse of various solvents to bring the solute into another liquid phase,precipitation, removal of impurities, dialysis, drying andrecrystallization, among others.

For the seperation and purification of the antibiotics from impurities,for example, its water soluble and basic properties are used, e.g.,while water soluble lower molecular impurities are not adsorbed, theantibiotics can be adsorbed on active carbon and eluted with anacidified aqueous alcohol and/or aqueous acetone.

The antibiotics cna be adsorbed strongly on cation exchange resins evenwhen they are in a state of neutral or weakly acid solution, and elutedwith a buffered basic solution, or an aqueous salt solution.

The aforementioned salts not only include the alkali salts of strongacids such as sodium chloride and ammonium chloride but also the alkalisalts of organic acids such as sodium acetate, and ammonium acetate, aswell as the salts of weak acids or weak bases such as those of aceticacid, and pyridine.

The antibiotics can be adsorbed weakly also on strong anion exchangeresins and eluted with water.

Among the ion exchange resins (strong or weak) which can be employed forthe aforesaid purpose, there may be mentioned, for example, AmberliteIR-100, 112 & 120, Amberlite XE-69, Amberlite IRC-50, Amberlite XE-89,Amberlite XE-64, Amberlite IR-45 & IRA-900 (All the above resins are theTrade Name of Rohm and Haas, Co.), Dowex-50Wx2, Dowex-1×2, Dowex-1×8(These three resins are the Trade Name of Dow Chemical, Co.), DuoliteCS-65 (Trade Name of Chemical Process, Co.), Permutit-50 (Trade Name isPermutit, Co.), etc. These resins may be prepared by such methods asdescribed in "Ion Exchange Resin" (Robert Kumin, Published by John Wiley& Sons, Inc., New York, N.Y., U.S.A. PP.).

The separation of the antibiotics are carried out by ion exchangechromatography using cation and anion exchange resins. For example, in atypical column chromatography for preparative purposes, the crudevalidamycins are chromatographed on Dowex 1×2 column (OH form, 100-200mesh, 870 ml.), and the column is developed with water to give the sixcomponents: Validoxylamines A and B and validamycins D, C, F and E, inorder of elution from the column.

Validamycin C-rich fractions and a fraction mainly containingvalidamycin D are rechromatographed on a silica gel column (elution withn-propanol, acetic acid, water 4:1:1) and Dowex 50w=2 column (elutionwith pyridine-acetic acid buffer, pH 6.0). Validamycins E and F areretarded on Dowex 1×2 column owing to their great affinity for theresin, these show a rather diffuse elution pattern on a gravity flow.The complete separation of validamycins E and F is not accomplished onDowex 1×2 column. Validamycin E-rich fractions and validamycin F-richfractions obtained by Dowex 1×2 chromatography are furtherchromatographed on Dowex 50W×2 column (elution with pyriding-acetic acidbuffer, pH 6.0). In this chromatography, early fractions containvalidamycin F. Thereafter, validamycin E is eluted.

Finally, chromatographically homogeneous validamycins C, D, E and F areobtained by rechromatography on Dowex 1×2 (OH form, developed withwater). Validoxylamine A is eluted just before validamycin D on Dowex1×2 column, and crystallization from water-ethanol give a purevalidoxylamine A. Although validoxylamine B and validamycin D are almostoverlapped on Dowex 1×2 chromatography, these components are easilyseparated on Dowex 50W×2 column(elution with pyridine-acetic acidbuffer, pH 6.0).

By using the foregoing procedures, validamycins C, D, E and F, andvalidoxylamines A and B are successfully isolated from a culture brothof Streptomyces hygroscopicus var. limoneus.

Each of validamycins C, D, E and F is obtained as a white powder andgives rise to validoxylamine A and D-glucose upon hydrolysis. However,those antibiotics are differ from one another in molecular structure,i.e. in the configuration of glycosidic bonds in the D-glucose moieties(α-or β-configuration) and in the positions and in the number ofD-glucose molecules.

The physicochemical properties of the validamycins C, D, E and F, andvalidoxylamines A and B are set forth below.

1. Structural formula

a. Validamycin C ##STR1## [R:O-α-D-glucopyranosyl-3-hydroxymethyl-4,5,6-trihydroxy-2-cyclohexenylgroup.]

b. Validamycin D ##STR2## [R₁ :O-α-D-glucopyranosyl-2,3,4-trihydroxy-5-hydroxy-methylcyclohexyl group.]

c. Validamycin E ##STR3## [R₂ : O-α-D-glucopyranosyl-Oβ-D-glucopyranosylgroup.]

d. Validamycin F ##STR4## [R₃ :O-α-D-glucopyranosyl-3-hydroxymethyl-4,5,6-trihydroxy-2-cyclohexenylgroup.]

e. Validoxylamine A ##STR5##

f. Validoxylamine B ##STR6## [R_(o) :2,3,4,6-tetrahydroxy-5-hydroxymethyl-cyclohexyl group.]

2. Melting or decomposition points

While none of validamycins C, D and E, and validoxylamines A and B has adefinite melting point, they are gradually decomposed at the followingtemperatures.

    ______________________________________                                        Validamycin C:     142°-160° C                                  Validamycin D:     125°-130° C                                  Validamycin E:     263°-268° C                                  Validamycin F:     165°-173° C                                  Validoxylamine A:  101°-105° C                                  Validoxylamine B:  102°-108° C                                  ______________________________________                                    

3. The elementary analysis, the molecular formulas as calculatedtherefrom, pKa' values, neutralization equivalents (molecular weight)and optical rotation values are given in Table 2.

                                      Table 2                                     __________________________________________________________________________    A table of the physicochemical properties of                                  validamycins C to F and validoxylamines A and B                                                                                        The number                                                                    of D-                                                         pKa'            glucose                    Estimated                   Optical                                                                              (neutralization                                                                         TLC Rf**                                                                            units and            Data  molecular                                                                             Molecular                                                                           Elementary analysis %                                                                       rotation                                                                             equivalent, in                                                                          (silica                                                                             their bond           Compound                                                                            formula  weight                                                                             Found  Calculated                                                                           [α]D(H.sub.2 O)                                                                aqueous solution)                                                                       gel G)                                                                              mode***              __________________________________________________________________________    Validoxyl-                                                                          C.sub.14 H.sub.25 NO.sub.8                                                            353.4 C 47.86                                                                              C 47.58                                                                              +170.0°                                                                       6.2 ± 0.2                                                                            0.34  --                   amine A                                                                             H.sub.2 O     H 7.86 H 7.70        (355 ± 30)                                      (335.4)*                                                                            N 3.89 N 3.96                                             B     C.sub.14 H.sub.25 NO.sub.9                                                            369.4 C 46.01                                                                              C 45.52                                                                              +172.4°                                                                       5.0 ± 0.2                                                                            0.42  --                                       H 7.28 H 7.36        (370 ± 30)                              H.sub.2 O                                                                             (351.4)*                                                                            N 3.59 N 3.79                                             Valida-                                                                             C.sub.26 H.sub.45 NO.sub.18                                                           677.7 C 46.48                                                                              C 46.08       6.0 ± 0.2                                                                            0.15  2                                  *     H 7.22 H 6.99        (680 ± 30)   α-and          mycin C                                                                             H.sub.2 O                                                                             (659.7)                                                                             N 2.15 N 2.07                        β-bonds         D     C.sub.20 H.sub.35 NO.sub.13                                                           515.5 C 46.97                                                                              C 46.60                                                                              +169.3°                                                                       6.0 ± 0.2                                                                            0.24  1                                  *     H 7.23 H 7.24        (520 ± 30)   α-bond               H.sub.2 O                                                                             (497.5)                                                                             N 2.58 N 2.72                                             E     C.sub.26 H.sub.45 NO.sub.18                                                           677.7 C 46.22                                                                              C 46.08                                                                              +148.2°                                                                       6.1 ± 0.2                                                                            0.15  2                                  *     H 7.19 H 7.02        (660 ± 30)   α-and                H.sub.2 O                                                                             (659.7)                                                                             N 1.94 N 2.07                        β-bonds         F     C.sub.26 H.sub.45 NO.sub.18                                                           677.7 C 46.00                                                                              C 46.08                                                                              +130.7°                                                                       6.1 ± 0.2                                                                            0.15  2                                  *     H 6.90 H 7.02        (680 ± 30)   α-and                H.sub.2 O                                                                             (659.7)                                                                             N 1.95 N 2.07                        β-bonds         __________________________________________________________________________     *The figure in parentheses denotes the empirical molecular weight as          calculated to the exclusion of H.sub.2 O.                                     **Developer solvent: n-propanol-acetic acid-water: 4/1/1.                     ***Estimated from the spin-spin coupling constant and optical rotation        value.   4. Ultraviolet absorption spectrum                              

When measured in an aqueous solution, validamycins C, D, C and F andvalidoxylamines A and B show no characteristic absorption maxima in theultraviolet region of the spectrum above 210 mμ excepting terminalabsorptions.

5. Infrared absorption spectrum (FIGS. l to 6)

The absorption spectra of validamycins C, D, E and F and validoxylaminesA and B as measured by the potassium bromide disc method are reproducedin FIGS. 1 to 6, respectively. It will be seen that their absorptionspectra are closely similar to one another. The dominant absorptions ofvalidamycins C, D, E and F, and validoxylamines A and B are as follows.

3400 (S), 2910 (M), 1638 (M), 1410 (M), 1075 (S), 1025 (S), 900 (M), 845(M) The abbreviations "M", "W" and "S" in parentheses denote mediumabsorptions, weak absorptions and strong absorptions.

6. Solubilities

Each of the antibiotics is readily soluble in water and soluble inpyridine, dimethylformamide and dimethylsulfoxide, except thatvalidoxylamine B is more sparingly soluble in pyridine.

In methanol, validamycin D and validoxylamine A are soluble butvalidamycins C, E and F, and validoxylamine B are more sparinglysoluble.

7. Color reactions

Each of the antibiotics gives positive reactions in periodicacid-benzidine and Greig-Leaback reagent (t-butylhypochlorite reagent)tests but gives negative reactions in Sakaguchi and Elson-Morgan tests.

Validamycins C, D, E and F, and validoxylamines A and B also givepositive reactions in anthrone, orcinsulfuric acid and naphthoresorcinolsulfuric acid tests.

8. Acid salt

The validamycins C, D, E and F have the weak base-property, forms a saltwith acid (e.g. hydrochloric acid, sulfuric acid, etc.). For example,the melting or decomposition points, elementary analysis of thehydrochloride of validamycins C, D, E and F are as following Table 3.

                  Table 3                                                         ______________________________________                                                    Decomposition                                                     Compound    points        Elementary analysis                                 ______________________________________                                        Validamycin C. HCl                                                                        115° - 132° C                                                                 C,    44.56                                                                              H,   6.32                                                          N,    1.53 Cl,  5.24                                Validamycin D. HCl                                                                        113° - 128° C                                                                 C,    44.61                                                                              H,   6.96                                                          N,    2.72 Cl,  6.74                                Validamycin E. HCl                                                                        141° - 157° C                                                                 C,    44.72                                                                              H,   6.54                                                          N,    1.72 Cl,  5.41                                Validamycin F. HCl                                                                        146° - 164° C                                                                 C,    44.83                                                                              H,   6.59                                                          N,    1.95 Cl,  5.32                                ______________________________________                                    

9. Ester

The hydroxyl groups of validamycins C, D, E and F can be esterified withan acid (e.g. acetic acid, malonic acid or malic acid). For example, themetling point or decomposition points, elementary analysis of theacetate of validamycins C, D, E and F are as following Table 4.

                  Table 4                                                         ______________________________________                                                      Decomposition                                                   Compound      point       Elementary analysis                                 ______________________________________                                        Acetyl validamycin C                                                                        98° - 105° C                                                                C, 51.45  H, 5.73                                                             N, 1.04                                             Acetyl validamycin D                                                                        94° - 106° C                                                                C, 52.86  H, 6.23                                                             N, 1.44                                             Acetyl validamycin E                                                                        96° - 107° C                                                                C, 51.57  H, 5.80                                                             N, 1.21                                             Acetyl validamycin F                                                                        103° - 108° C                                                               C, 51.48  H, 5.89                                                             N, 1.26                                             ______________________________________                                    

10. Acute toxicity of the antibiotics

The toxicity of the antibiotics can be confirmed, for instance, byadministering each of the validamycins C, D, E and F, andvalidoxylamines A and B, intravenously to mice and measuring the LD₅₀value, or by measuring the median lethal dose (48 hours) for Oryziaslatipes. The results of such measurements are shown in Table 5.

                                      Table 5                                     __________________________________________________________________________    Acute toxicity                                                                Test animal                                                                           Antibiotics                                                                            Test procedure                                                                           Toxicity                                          __________________________________________________________________________           Validamycin C     LD.sub.50 > 2000 mg/kg                                      Validamycin D     LD.sub.50 > 2000 mg/kg                                      Validamycin E                                                                           Intravenous                                                                           LD.sub.50 > 2000 mg/kg                               Mice                                                                                 Validamycin F                                                                           injection                                                                             LD.sub.50 > 2000 mg/kg                                      Validoxylamine A  LD.sub.50 > 2000 mg/kg                                      Validoxylamine B  LD.sub.50 > 2000 mg/kg                                      Validamycin C     TL.sub.m > 1000 ppm                                         Validamycin D     TL.sub.m > 1000 ppm                                  Oryzias                                                                              Validamycin E                                                                           Leaving in                                                                            TL.sub.m > 1000 ppm                                  latipes                                                                              Validamycin F                                                                           water solution                                                                        TL.sub.m > 1000 ppm                                         Validoxylamine A  TL.sub.m > 1000 ppm                                         Validoxylamine B  TL.sub.m > 1000 ppm                                  __________________________________________________________________________

The biological properties

When a 1 mg./ml. aqueous solution of each of validamycins C, D, E and Fand validoxylamines A and B is used as a stock solution, the highestdilution factor that causes an abnormal branching at the hyphal tips ofPellicularia sasakii is as follows.

    ______________________________________                                        Validoxylamin A    100                                                        Validoxylamine B    10                                                        Validamycin C      100                                                        Validamycin D       10                                                        Validamycin E      70,000 - 80,000                                            Validamycin F      70,000 - 80,000                                            ______________________________________                                    

Thus obtained validamycins C, D, E and F and validoxylamines A and B canbe employed for combating the plant diseases. For the purpose, they areused in the form containing the cultured broth of a validamycin-groupproducing strain belonging to the genus Streptomyces, filtrate thereof,the concentrate thereof, or a purified preparation thereof. It is alsopossible to employ validamycins C, D, E and F and validoxylamines A andB in the form of a free base or as the salts with suitable organic orinorganic acid (i.e. oxalic acid, succinic acid, sulfuric acid,hydrochloric acid, etc.) or as complexes with metals (e.g. sodium,cobalt, copper, aluminum, calcium, etc.). It is further permissible toemploy an ester of the antibiotics, for instance, which may be obtainedby esterifying the hydroxyl of the antibiotics with an acid (e.g. aceticacid, malonic acid or malic acid).

These compositions can be applied in any suitable manner. Thus,depending upon the purpose of application, application time, applicationmethod, etc., the composition may be directly applied as such, afterdissolution or dispersion in a suitable liquid carrier, or afterblending with a suitable solid carrier. If desired, by adding anemulsifying agent, dispersant, suspending agent, adsorbent, penetrant,wetting agent, thickener, stabilizer or/and adjuvant, the presentcomposition may be used in such varied formulations as an oil solution,emulsion, wettable powder, aqueous solution, soluble powder, dust,tablet, granule and spray, etc.

It is further allowable to use such a composition after compounding orblending with such other germicides as copper germicides, organic sulfurgermicides, organic chlorine germicides, organic phosphorus germicides,other antibiotics, etc., such insecticides as organic chlorineinsecticides, organic phosphorous insecticides, carbamic acidinsecticides, natural insecticides, etc., ascaricides, nematocides,herbicides, plant growth regulators, synergistic agents, attractants,repellents, perfumes, plant nutrients, fertilizers and the like.

A concentration of the active ingredient in the present fungicides readyfor use is usually from about 0.0001% to about 0.5% by weight, morepreferably about 0.0003% to about 0.3% by weight, in case of the liquidform (i.e. a solution, a suspension or an emulsion); while in case ofthe solid composition, from about 0.01% to about 30% by weight, about0.1% to about 20% by weight is preferable. But, upon necessity, acomposition containing at a higher or lower concentration than theabove-mentioned value may be put into use. The content of the activeingredient of composition may be in from about 0.5% to about 80% byweight in relative to the composition, when it is prepared for aconcentrate form.

Some formulations of the fungicide containing validamycins C, D, E and Fand validoxylamines A and B are illustrated as follows:

                  Formulation 1                                                   ______________________________________                                        Wettable powder                                                               Validamycin C           1.0%                                                  Sodium ligninsulfonate  0.1%                                                  Polyoxyethylene nonylphenyl ether                                                                     0.1%                                                  White carbon            0.1%                                                  Clay                    98.7%                                                 ______________________________________                                    

Depending upon the purpose and method of application, the aboveformulation is diluted with water to the range of 2-400 ppm in terms ofthe present antibiotic and the diluted preparation is sprayed by meansof a sprayer, applied to the soil surface, or the above powder is usedundilutedly to coat the seed.

                  Formulation 2                                                   ______________________________________                                        Tablets                                                                       Validamycin E           15.0%                                                 Polyoxyethylene nonylphenyl ether                                                                     2.0%                                                  Lactose                 83.0%                                                 ______________________________________                                    

Before application, the above formulation is dissolved in water to makethe concentration range shown in Formulation 1.

                  Formulation 3                                                   ______________________________________                                        Aqueous solution                                                                      Validamycin D                                                                             40.0%                                                             Methanol    5.0%                                                              Aluminum stearate                                                                         5.0%                                                              Water       50.0%                                                     ______________________________________                                    

The above formulation is diluted with water to the range of1,000-200,000 ppm. and the diluted preparation is sprayed by means of afine concentrate sprayer from aircraft, for instance.

                  Formulation 4                                                   ______________________________________                                        Emulsifiable concentrate                                                      Validamycin F           10.0%                                                 Polyoxyethylene nonylphenyl ether                                                                     5.0%                                                  Methanol                20.0%                                                 Methylnaphthalene       40.0%                                                 Dimethylformamide       25.0%                                                 ______________________________________                                    

Before application, the above formulation is diluted with water to theconcentration range given in Formulation 1.

                  Formulation 5                                                   ______________________________________                                        Dust                                                                          Validoxylamine A  3.0%                                                        Aluminum stearate 0.02%                                                       Talc              96.98%                                                      ______________________________________                                    

Depending upon the objective and mode of application, the above mixtureas such is dusted by means of a duster at the rate of 1-6 kg. to 10 are,or used to coat the seed.

                  Formulation 6                                                   ______________________________________                                        Mixed dust A                                                                  Validamycin E     4.0%                                                        Validamycin F     1.0%                                                        Aluminum stearate 0.02%                                                       Talc              94.98%                                                      ______________________________________                                    

The above preparation is directly applied at the rate of 1-50% per seedsweight to coat the seed.

EXPERIMENT 1

Rice sheath blight control test

(Test procedure)

Test plants:

Rice Plants (variety: Kinmaze) are planted in earthen pots 9 cm indiameter, 3 stocks per pot. Seedlings 80 days after potting are testedand 6 pots each of them are grouped.

Inoculating method:

Pathogenic fungi Pellicularia sasakii is cultivated on a plate of potatoinfusion-sugar-agar at 30° C for 48 hours, and its agar disc 10mm indiameter is cut out from a peripheral colony. The disc is inserted intothe inside space of the leaf sheath near the soil surface, a disc perstem. The pots are kept in a PVC shelter at the temperature of 32°-35° Cand the relative humidity of 100-70%. Inoculation is carried outimmediately after the applied drug has been air-dried in theprophylactic test, and 3 days before application in the remedial test.

Application of the drug:

Each of test drugs is diluted in water to prepare the fixedconcentration of the active ingedient, as shown in Table 3, and theaqueous solution is sprayed evenly over the foliage by means of aspray-gun at the rate of 30 ml. per 6 pots.

Assay method:

10 days after application of the drug, the length of each stem fromground level to the upper edge of the lesion is directly measured.

                                      Table 6                                     __________________________________________________________________________    Leaf blight control effect                                                    (The average length of lesions/stem(cm)                                       __________________________________________________________________________               Preventive effect.sup.a (%)                                                                    Remedial effect.sup.a (%)                                                                         Injury                           Concentration                                to plant                      drug                                                                             of drug (ppm)                                                                          3 10 30 100 300 1  3  10 30 100 300 (300 ppm)                     __________________________________________________________________________    Validamycin C                                                                            46 34 4  1   0   -  68 22 12 10  7   -                             Validamycin D                                                                            28 5  1  0   0   -  -  99 20 11  8   -                             Validamycin E                                                                             3 2  0  0   0   41 14 12 11  5  3   -                             Validamycin F                                                                            21 2  0  0   0   27 13 13 12 12  2   -                             Validamycin E + F.sup.b                                                                   1 0  0  0   0   9  5   3  2  1  1   -                             Validoxylamine A                                                                         28 4  1  0   0   -  83 28 14 10  7   -                             Validoxylamine B                                                                         49 47 32 16  8   -  -  93 75 51  10  -                              MAFA.sup.c                                                                              76 51 0  0   0   -  89 30 15 13  10  +                             __________________________________________________________________________     (Note)                                                                        .sup.a Percent of the average length of lesion/stem(cm) per control           untreated (34.4cm)                                                            .sup.b Mixture of validamycins E (80%) and F (20%)                            .sup.c Methyl arsonic acid ammonium complex salt                         

EXPERIMENT 2

Cucumber damping-off control test

(Test procedure)

Rhyzoctonia solani is cultured on barley medium at 28° C for 5 days. Afield soil sample is packed into clay pots 9 cm in diameter, which arethen sterilized with steam. The chaff inoculum is evenly worked into thecover soil of each pot to the depth of about 3 cm, at the rate of2g./pot. The pots are kept in an incubation chamber at 28° C for 4 daysand, then, transferred to a greenhouse.

Application of the drug:

As per Experiment 1, the drug is sprayed onto the soil surface at therate of 30 ml./6 pots/group.

Test plant:

Healthy seeds of cucumber (variety: Yonyo) are sown by burial in thepots immediately after treatment, at the rate of 10 seeds/pot. The potsare then placed in a greenhouse at 32°C-28° C.

Assay method:

14 days after sowing, the results are evaluated against the followingcoefficients of lesion and the degrees of damage are calculated.

Coefficients of lesion (I)

O : Healthy

0.5 : Root hairs alone are slightly affected. 1 : Aerial portions nearsoil surface and roots affected.

2 : Early stage of damping-off.

3 : Affected at germination; growth inhibited. ##EQU1## wherein n meansthe number of samples corresponding to each coefficient of lesion and Nmeans the total number of plants.

                  Table 7                                                         ______________________________________                                        Damping-off control effect                                                                 Concentration                                                                              Degree of                                                        of drug      damage    Injury                                    Drug         (ppm)        (%)       to plant                                  ______________________________________                                        Untreated control                                                                          -            100       -                                         Validamycin C                                                                              5            100       -                                                      20            84       -                                                      80           0         -                                         Validamycin D                                                                              5            100       -                                                      20            93       -                                                      80           0         -                                         Validamycin E                                                                              5             10       -                                                      20           0         -                                                      80           0         -                                         Validamycin F                                                                              5             80       -                                                      20           0         -                                                      80           0         -                                         Validamycin(E+F)                                                                           5(4+1)       7         -                                                      20(16+4)     0         -                                                      80(64+16)    0         -                                         Validoxylamine A                                                                           5             84       -                                                      20           6                                                                80           0         -                                         Validoxylamine B                                                                           5            100       -                                                      20           100       -                                                      80            75       -                                         Pentachloro- 5            100       -                                         nitrobenzene 20           100       -                                                      80           100       -                                         ______________________________________                                    

EXPERIMENT 3

Tomato leaf mold control test

(Test procedure)

Test plants:

Tomato plants (variety: ponterosa) are planted in 12 cm in diameterpots, one plant per pot. The seedlings 50 days after potting are used, 6pots/group.

Application of the drug:

Applied at the rate of 100 ml./6 pots/group as per Experiment 1.

Inoculating method:

Pathogenic fungi Cladosporium fulvum is cultured on a potatoinfusion-sucrose plate at 20° C for 14 days, and a suspension of conidia(5×10⁵ /ml.) is prepared from the culture.

The plants are spray-inoculated with the suspension 3 days beforeapplication of the drug, at the rate of 5 ml. per pot. The pots are keptin an inoculation chamber at 25° C for 24 hours, and, then they aretransferred to a greenhouse.

Assay method:

14 days after inoculation, the specific ratio of the average area oflesion in the leaves is measured.

(Result)

                  Table 8                                                         ______________________________________                                        Tomato leaf mold control effect                                                            Concentration                                                                            Specific area                                                      of drug    of lesion  Injury to                                  Drug         (ppm)      (%)        plant                                      ______________________________________                                        Untreated    -          98         -                                          control                                                                       Validamycin C                                                                              50         18         -                                          Validamycin D                                                                              50         25         -                                          Validamycin E                                                                              50          2         -                                          Validamycin F                                                                              50          3         -                                          Validamycin (E+F).sup.a                                                       Validoxylamine A                                                                           50          6         -                                          Validoxylamine B                                                                           50         36         -                                          ______________________________________                                         .sup.a Mixture of validamycins E (80%) and F (20%).                      

The followings are examples for production of the antibiotics and theliquid-chromatographic equipment to be used for analysis in theseexamples are as follows. For separation and feeding, the JLC-BC₂equipment manufactured by Japan Elctronics Co., Ltd. is employed.

For the detection of active compounds giving a negative orcin-sulfuricacid test, the differential refractometer model R 403 (Water Associates,Inc.) is utilized.

The following conditions were employed.

(Analysis I)

Analytical column:

A column (70 cm × 8 mm I.D.) packed with the resin AG 1 × 2 (200-400mesh, OH-form, BIO-RAD-laboratory).

Eluant;

water: flow rate: 0.37 ml./min.

(Analysis II)

Analytical column:

a column (15 cm × 8 mm I.D.) packed with Jeol Resin LCR-3 (JapanElectronics, sulfate-form ion exchange resin).

Eluant:

(i) 0.25 M borate buffer (pH 9.0); flow rate: 0.49 ml./min.; (ii) 0.15 Mborate buffer (pH 7.7); flow rate: 0.49 ml./min. To detect the compoundsgiving negative orcin-sulfuric acid tests, the differentialrefractometer of Water Associates, for instance, was employed.

Gas chromatographic measurements were made using Hitachi GasChromatograph Model 063 (Hitachi, Ltd.) and Yanaco Gas chromatographModel 5DH (Yanagimoto Co., Ltd.).

The samples were prepared as described below. A dry sample (a few mg.)containing the components of the validamycin complex was dissolved indry pyridine (0.5 ml.) and, as an internal reference, melezitose (0.5mg.) was added to the solution. Then, trimethylchlorosilane (0.5 ml.)and bis-(trimethylsilyl)-acetamide (0.5 ml.) were further added. Themixture was allowed to react at 70° C for 20 minutes and the reactionproduct was injected into the gas chromatographic column.

As regards the column, good separations can be obtained if Chromosolb WAW DMCS (Johns-Manville, Co.) or Chromosolb G AW DMCS (Johns-Manville,Co.), for instance, is used as the stationary phase, and SiliconOV-1,OV-17 (1-5%) (Ohio Valley Speciality Chemical Co.) for instance, isused as the liquid phase in a glass column (1-2 m). The temperature ofthe column is maintained at 250°-300° C.

EXAMPLE 1

To an aqueous solution (adjusted to pH 7) containing 3% of glucose, 2.2%of raw soybean flour and 0.3% of peptone is added 0.4% of precipitatedcalcium carbonate, and 2 liters of the solution is despensed into 4flasks of 2 liters capacity, 500 ml. per flask. After sterilization,each of the flasks is inoculated with a loopful of Streptomyceshygroscopicus var. limoneus (ATCC No: 21431) from its slant culture. Theflask is incubated on a reciprocating shaker at 28° C for 2 days toprepare a seed culture.

Separately, a stainless-steel tank of 200 liter capacity is charged with100 liters of a medium (pH 7.0) composed of an aqueous solutioncontaining 5% glucose, 3.6% raw soybean flour and 0.5% peptone, and 0.6%precipitated calcium carbonate. After sterilization, the tank isinoculated with 2 liters of the above seed culture and incubated for 114hours. The procedure yields 68 liters of a culture filtrate containing10,000 dilution units/ml. of the validamycin complex.

EXAMPLE 2

A culture (950-liters)of Streptomyces hygroscopicus var. limoneus,(ATCC:21431), which is obtainable by a procedure similar to thatdescribed in Example 1, is adjusted to pH about 4.0 with oxalic acidand, with the addition of a filtration aid, filtered. The resultingfiltrate (720-liters) is passed through a column (resin: content120-liters) of Amberlite (Rohm & Haas Company) IR-120 (OH-form). Thiscolumn is washed with water (240-liters), then the washings are combinedwith the effluent. The pool is passed through a column (resin content:140-liters) of Amberlite (same as above) IR-45 (OH-form) and, then, runonto a column (resin content: 60-liters) of Dowex (Dow Chemical Company)50W × 2 (H-form). The latter column is washed with water and eluted with0.5N-aqueous ammonia. The active fractions are pooled and concentratedunder reduced pressure.

The concentrate (40-liters) is chromatographed using a column (resincontent: 30-liters) of Amberlite IRA-900 (OH-form) and, as an eluant,water. Each fraction is assayed by thin-layer chromatography (silica gelG; developer: n-propanol-acetic acid-water: 4/1/1), gas chromatographyand liquid chromatography. The active compounds are eluted in the orderof validoxylamines A and B, and validamycins D, C, F and E. Eachfraction is concentrated to the consistency of a syrup, followed by theaddition of acetone. The prcedure yields 1.1 kg. of total of a crudepowder of validamycin complex.

EXAMPLE 3

Each of the fractions containing validamycins C, D, E and F andvalidoxylamines A and B eluting from Amberlite IRA-900 in Example 2 isconcentrated under reduced pressure to the consistency of a syrup,followed by the addition of acetone to precipitate the activecomponents. The procedure yields 1.1 kg. of total of a crude powder ofvalidamycin complex. In 2 liters of water is dissolved 400 g. of theabove crude powder and the solution is chromatographed using a column(resin content: 22-liters) of Dowex 1 × 2 (OH-form) and, as an eluant,water. Each of the fractions is assayed by thin-layer chromatography andgas chromatography. Validoxylamines A, B and validamycin D are found inthe 25 to 55-liter fractions, validamycin A in the 75 to 115-literfractions, validamycins B and C in the 135 to 275-liter fractions adnvalidamycins E and F in the 325 to 760-liter fraction. Each group offractions is collected and concentrated under reduced pressure, followedby adding acetone to precipitate white powders. The procedure gives 42.8g. of a white powder which is predominantly composed of validamycin D,179.2 g. of a white powder composed predominantly of validamycin A, 42.0g. of a white powder composed mainly of validamycins B and C, and 5.6 g.of a white powder mainly containing validamycins E and F.

EXAMPLE 4

Ten grams of the crude product relatively rich in validamycin D, whichis obtainable according to Example 3, is chromatographed using a column(700 ml.) of Dowex 1 × 2 (OH-form, 100-200 mesh). The eluate is scannedwith a differential refractometer and each of the fractions isidentified and tested for purity by gas chromatography. It is found thatvalidoxylamine A, in 0.8 to 2.0 liter fractions validoxylamine B andvalidamycin D occurs in the 1.6 to 2.0 liter fractions, respectively.The eluate containing validoxylamine A is concentrated to dryness underreduced pressure to obtain about 2.0 g. of a white powder. The eluatecontaining validamycin D and validoxylamine B are concentrated todryness under reduced pressure to obtain about 4.6 g. of a white powder.

The above powder is dissolved in 0.1M-pyridine-acetate buffer (pH 6.0)and the solution is run onto a column (700 ml.) of Dowex 50W × 2buffered with the same buffer as above. The column is eluted with asimilar buffer, whereupon validoxylamine B and validamycin D are elutedin the 1.5 to 2.5 liter fractions and the 7.5 to 8.5 liter fractions,respectively. Each fraction is concentrated under reduced pressure andthe concentrate is purified using a column (100 ml.) of Dowex 1 × 2(OH-form) (eluant: water).

The eluate is concentrated under reduced pressure and the concentrate isadded acetone to precipitate 0.7 g. of a white powder of validoxylamineB and 3.2 g. of a white powder composed of validamycin D.

To obtain a high purity grade of validamycin D, the crude productobtained is purified by chromatography on silica gel (Merck's silica gel0.05-0.20 mm; n-propanol-acetic acid-water:4/1/1) and Dowex 50W × 8(eluant: 0.1M pyridine-acetate buffer, pH 6.0). Finally, the product isfurther purified with Dowex 1 × 2 (OH-form; eluant: water).

When hydrolyzed with acid, validamycin D yields 1 mole each ofvalidoxylamine A and D-glucose.

EXAMPLE 5

Fifteen (15) grams of the crude product relatively rich in validamycinsB and C, which is obtainable according to Example 3, is chromatographedusing a column (870 ml., 3.5 cm × 95 cm) of Dowex 1 × 2 (OH-form,100-200 mesh) and, as an eluant, water. Each of the fractions is testedby thin-layer chromatography (silica gel G; n-propanol-acetic acid;water: 4/1/1; color reagent; naphthoresorcinol or gas chromatography.Validamycin A (yield: about 5.0 g.) is eluted in the eluatecorresponding to about 5 to 7 times of the volume of resin; validamycinC (yield: about 0.2 g.) in the eluate corresponding to about 10 to 20times of the volume of resin; and validamycin B (yield: about 6.0 g.) inthe eluate corresponding to about 12 to 18 times the volume of resin.

Since the crude powder obtained by the above-described procedure ispredominantly composed of validamycin C but still contains a minoramount of validamycin B, it is further purified by the followingprocedure.

One (1.0) gram of the above powder is chromatographed using a column(1.3 cm × 90 mm; 200 ml.) of silica gel (Merck's silica gel, 0.05-0.2mm) and, as a developer, n-propanol-acetic acid-water (4:1:1). Of theresulting fractions, those which give positive naphthorescorcinolreactions are confirmed by thin-layer chromatography. The fractionswhich contain validamycin C alone are collected and concentrated todryness. The residue is further absorbed on Dowex 50W ×2(pyridine-form), which is eluted with 1N-pyridine-acetate buffer (pH6.5). The fractions giving a positive naphthorescorcinol test arecollected and concentrated to dryness under reduced pressure, whereupona white powder of validamycin C is obtained. For the purpose ofobtaining physicochemical data, the powder is further purified by meansof a column of Dowex 1 × 2 (OH-form).

When hydrolyzed with acid, validamycin C yields 1 mole of validoxylamineand 2 moles of D-glucose.

EXAMPLE 6

The crude product (5.2 g.) obtained from the fractions comparativelyrich in validamycins E and F, which are obtainable according to Example3, is chromatographed using a column (2.7 cm × 43 cm) of Dowex 1 × 2(OH-form, 100-200 mesh) and, as an eluant, water. The eluate isfractionated with a refractive index detector and each fraction isidentified and tested for purity by means of automatic liquidchromatography and gas chromatography.

Validamycin B is eluted with distilled water in the eluate correspondingto about 20 times of the volume of the resin and validamycins E and Fare obtained in the eluate corresponding to about 30 times of the volumeof the resin.

In this procedure, validamycin F is eluated somewhat ahead ofvalidamycin E and, as determined by automatic liquid chromatography, thefirst fractions contain comparatively large amounts of validamycin F andthe later fractions rich in validamycin E. The fractions containing 80%or more validamycin E are concentrated to dryness under reducedpressure, whereupon 1.3 g. of a white powder is obtained.

The resulting powder containing 80% or more validamycin E is purified bychromatography on Dowex 50W × 8 (pyridine-form; eluant: 0.1Mpyridine-acetate buffer, pH 6.0) and, then, on Dowex 1 × 2 (OH-form;eluant: water). The fractions containing validamycin E are collected andconcentrated to dryness under reduced pressure. The procedure yields awhite powder of validamycin E. When necessary, the above procedure isrepeated once, whereupon a pure grade of validamycin E is obtained.

When hydrolyzed with acid, validamycin E yields 1 mole of validoxylamineand 2 moles of D-glucose.

In the same manner as described for validamycin E, the fractions rich invalidamycin F are collected and purified to obtain a white powder ofvalidamycin F. When necessary, the same procedure is repeated once,whereupon a pure grade of validamycin F is obtained.

When hydrolyzed with acid, validamycin F yields 1 mole of validoxylamineA and 2 moles of D-glucose.

Though validamycins E and F have similar physicochemical properties, aliquid chromatographic study reveals a significant difference betweenthe two antibiotics; e.g. while validamycin F shows a retention time of197 minutes, validamycin E shows a retention time of 233 minutes.

Incidentally, under the same conditions, validamycin C shows a retentiontime of 146 minutes.

EXAMPLE 7

To 150 mg. of the validamycin C obtained according to Example 5 areadded 1 ml. of pyridine and 1 ml. of acetic anhydride, and the mixtureis reacted and dissolved on a water bath under heating and stirring. Thereaction mixture is allowed to stand at room temperature overnight,followed by the addition of water. The resulting precipitate isextracted with chloroform and the extract is washed with water. Thechloroform is distilled off and water is added to the residue, whereupon258 mg. of acetyl-validamycin C is obtained. [α]_(D) ²³ + 98.9° (C=1.0,CHCl₃);

Elementary analysis:

Found C, 51.45; H, 5.73; N, 1.04;

Molecular weight: 1245 ± 50 (VPO**method, in ethyl acetate).

The validamycin C acetate thus obtained (350 mg.) is dissolved in 3.5ml. of methanol, and 20 ml. of sodium methylante (a solution of 0.5 g.sodium metal in 100 ml. methanol) is added. The mixture is allowed tostand at room temperature for 5 hours, at the end of which time it isrun onto a column of Dowex 50W × 8 (H-form). The column is washed withwater and, then, eluted with 1 N-HC1. The eluate is furtherchromatographed using a column of Dowex 1 × 2 (OH-form; eluant: water)and the active fractions are concetrated to dryness under reducedpressure to recover validamycin C.

EXAMPLE 8

To 400 mg. of the validamycin D obtained according to Example 4 areadded 2.5 ml. of pyridine and 2 ml. of acetic anhydride, and the mixtureis allowed to stand at room temperature overnight. It is treated in thesame manner as Example 7 to obtain 690 mg. of acetyl-validamycin D. Thisproduct gives the following analytical data. [α]_(D) ²³ + 145° (C=1.0,CHCl₃);

Elementary analysis:

Found C, 52.86; H, 6.23; N, 1.44;

Molecular weight: 960 ± 50 (VPO method, in ethyl acetate).

At O° C, 30 ml. of methanol saturated with ammonia is added to the aboveacetyl-validamycin D (550 mg.), and the mixture is allowed to stand atroom temperature overnight. The reaction mixture is concentrated underreduced pressure and, then methanol is added to the residue. Thisprocedure is repeated three times. The final residue is dissolved inwater and purified by means of a column of Dowex 1 × 2 (OH-form),whereupon 228 mg. of validamycin D is obtained.

EXAMPLE 9

In N,N-dimethylformamide (5 ml.) is dissolved 1.37 g. of the crudeproduct containing validoxylamine A (purity: about 80%) and the solutionis refluxed with 10 ml. of acetone dimethyl ketal (2,2-dimethoxypropane)and 0.65 g. of p-toluenesulfonic acid in a water bath for 5 hours. Thereaction mixture is allowed to cool and, then, the excess acetonedimethyl ketal is removed by distillation. The residue is dissolved inchloroform and, after washing with water, the chloroform layer isconcentrated. The concentrate is purified by chromatography on silicagel (2 cm × 25 cm; eluant: chloroform) and the fractions are collectedand concentrated. Then, ethanol is added to the concentrate to obtaincrystals. m.p.228°-231° C (decomp.); [α]_(D) ²³ + 97.0° (c=1.0, CHCl₃);

Elementary analysis:

Found: C, 62.56; H, 8.24; N, 2.83.

500 mg. of the isopropylidene-validoxylamine A thus obtained is refluxedtogether with 50 ml. of a 50% aqueous solution of acetic acid for about5 hours until a clear solution is obtained. This solution isconcentrated to dryness and, if required, purified by chromatography onDowex 1 × 2 (OH-form). The product is finally crystallized fromwater-ethanol. Yield 285 mg.

What is claimed is:
 1. A pure antibiotic selected from the groupconsisting ofa. pure validamycin C of the formula ##STR7## wherein R isO-α-D-glucopyranosyl-3-hydroxymethyl-4,5,6-trihydroxy-2-cyclohexenyl,and decomposing at 142° to 160° C, b. pure validamycin D of the formula##STR8## wherein R₁ isO-α-D-glucopyranosyl-2,3,4-trihydroxy-5-hydroxy-methyl-cyclohexyl, andhaving the optical rotation [α]_(D) (H₂ 0) = +169.3°, c. purevalidamycin E of the formula ##STR9## wherein R₂ isO-α-D-glucopyranosyl-O-β-D-glucopyranosyl, and having the opticalrotation of [α]_(D) (H₂ O) = +148.2°, d. pure validamycin F of theformula ##STR10## wherein R₃ isO-α-D-glucopyranosyl-3-hydroxymethyl-4,5,6-trihydroxy-2-cyclohexenyl,and having the optical rotation [α]_(D) (H₂ 0) = +130.7° and decomposingat 165° to 173° C,and acid salts of each of validamycin C, D, E and F.2. A pure antibiotic according to claim 1, wherein the antibiotic is ahydrochloride salt.
 3. A pure antibiotic according to claim 1, whereinthe antibiotic is a sulfate salt.
 4. A pure antibiotic according toclaim 1, wherein the antibiotic is an oxalate salt.
 5. A pure antibioticaccording to claim 1, wherein the antibiotic is a succinate salt.